Oligonucleotide technology utilizes short sequences of nucleic acids as highly specific probes to assay the presence of DNA and/or RNA sequences of interest. It also has applications for treating diseases as it can be used to selectively inhibit the expression of genes of interest. As such, oligonucleotide technology has great potential in the analysis and treatment of genetic disorders.
As an example, genetic disorders account for approximately 30 percent of admissions to pediatric hospitals. Therefore, the ability to provide a comprehensive diagnostic molecular genetic service is an important aspect of a modern health care system.
In order to solve problems attendant in some applications using oligonucleotides, such as degradation by host cell nucleases, or transfer across cell membranes, oligonucleotide analogs have been constructed by replacing a non-bridging oxygen in the phosphate moiety with a sulfur, methyl or ethyl group (reviewed in Stein, C. A., and Cheng, Y.-C., Science (1993) 261: 1004). A second example of an oligonucleotide analog is one using a formacetal/ketal type linkage replacing one or more phosphodiester linkages between adjacent nucleosides. (U.S. Pat. No. 5,264,564, issued Nov. 23, 1993).
Dimeric nucleosides have also been constructed with uncharged guanidyl linkages replacing some phosphodiester bonds. Vandendriessche et al. provide a method for synthesizing thymidine nucleosides coupled together with substituted guanidyl linkages, J. Chem. Soc. Trans. 1: 1567 (1993); Bioorg. Med. Chem. Lttrs. 3(2): 193-198 (1993). The electron-withdrawing properties of the substituent groups make the guanidyl moiety uncharged at physiological Ph. These thymidine dimers can be incorporated into oligonucleotide chains with standard phosphate backbones.
Vandendriessche et al. developed their method using neutral guanidyl groups for coupling nucleosides because they reasoned that positively charged groups may hamper cellular uptake of oligonucleosides containing them and may lead to non-specific binding.
However, the resulting method having uncharged guanidyl units at physiological pH is limited in its applications to oligonucleoside technology. It does not allow for construction of oligonucleoside chains containing two or more successive guanidyl linkages. Instead, each nucleoside connected to one nucleoside via a guanidyl linkage must be attached to a second with a phosphodiester linkage. Thus, many of the advantages provided by guanidyl linkages are undermined.
For example, the necessity for phosphodiester bonds following guanidyl bonds exposes oligonucleosides to cellular nucleases that recognize phosphodiester bonds.
A second limitation of the Vandendriessche method is that the electrically neutral guanidyl bridges will not interact with the phosphates on the backbones of naturally occurring nucleic acids.
The present invention overcomes both of these shortcomings. First, it provides an alternative approach to constructing oligonucleosides that utilizes guanidyl linkages having a positive charge at physiological pH. In addition, the method provides a way to construct a oligonucleoside chain built with successive positively charged guanidyl linkages. It is thus possible to use the present invention to construct a positively charged oligonucleoside chain that will bind with high affinity to negatively charged phosphate backbone of cellular and viral DNA or RNA.
The higher affinity binding reduces the incidence of false positives and facilitates earlier detection of a diseased state. Early detection of disease improves the patient's prognosis and aids in determining an optimal therapy regimen. Therefore, the diagnostic agents of the invention provide a means to determine disease states early, efficiently and predictably.